RICE BLAST SUSCEPTIBILITY GENE Pi21, RESISTANCE GENE pi21, AND USES THEREOF

ABSTRACT

The present inventors succeeded in isolating the rice field resistance gene pi21 by linkage analysis, and found that field resistance to blast in plants could be modified by introducing or controlling the expression of the gene. Thus, it became possible to efficiently confer plants with field resistance. It also became possible to select, at an early stage, rice plants having field resistance to blast. Moreover, by changing the tissue specificity of expression and the expression level of the gene involved in field resistance, varieties having resistance as well as high practical use can be grown.

RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 12/881,685, filed Sep. 14, 2010, which is a divisional applicationof U.S. application Ser. No. 11/922,994, filed Apr. 24, 2008, which is a35 U.S.C. 371 national stage filing of International Application No.PCT/JP2006/0311341, filed Jun. 6, 2006, which claims priority toJapanese Application No. 2005-187867, filed Jun. 28, 2005. The entirecontents of each of these applications are hereby incorporated byreference herein.

TECHNICAL FIELD

The present invention relates to pi21, a gene conferring rice with blastfield resistance, and methods for modifying field resistance to blast inplants by using the gene.

BACKGROUND ART

Resistance of rice to blast fungi is classified into two types: trueresistance and field resistance (Non-patent Document 1). The former isbased on hypersensitive reactions, and is a very effective andqualitative resistance highly specific to race. It has been known byexperience that a variety introduced with a single resistance gene losesits effect in a few years due to the appearance of fungi compatible withthe gene. On the other hand, field resistance is defined as thedifference of resistance among varieties that is observed underconditions where true resistance is not functioning. Although the effectof field resistance is smaller compared to true resistance, it ispractically useful because it has low race specificity and can confercontinuous resistance to varieties.

Thirty or more kinds of genes associated with true resistance are known,and of these, Pib and Pita genes have been isolated (Non-patent Document2). It has been found that these genes are NBS-LRR class genes havingnucleotide binding sites (NBS) and leucine-rich repeats (LRRs), withstructures similar to previously reported plant disease resistancegenes. Like other disease resistance genes, products of plant resistancegenes are considered to have a receptor function, directly or indirectlyrecognizing products of nonpathogenic genes in pathogens correspondingto the diseases. It has been actually revealed that Pita physically anddirectly binds to a nonpathogenic gene product.

As for field resistance, Japanese upland rice varieties are known tohave excellent traits, and chromosomal positions of multiple gene lociinvolved in field resistance have been identified (Non-patent Document3). However, the structure and expression mechanisms of the genes havenot been elucidated, and thus field resistance cannot yet be efficientlyused for breeding selection compared to true resistance. Multiplechromosome regions in the West Africa upland rice variety Moroberekanhave been reported to play a role in incomplete resistance, which is aconcept similar to field resistance (Non-patent Document 4); however, nogenes have been identified.

-   [Patent Document 1] Japanese Patent Application Kokai Publication    No. (JP-A) 2000-93028 (unexamined, published Japanese patent    application)-   [Patent Document 2] JP-A 2000-342262-   [Patent Document 3] (Granted/Registered) Japanese Patent No.3376453    (P3376453)-   [Patent Document 4] JP-A 2003-88379 (P2003-88379A)-   [Patent Document 5] JP-A 2003-199577 (P2003-199577A)-   [Patent Document 6] JP-A 2003-199448 (P2003-199448A)-   [Patent Document 7] JP-A 2004-329215 (P2004-329215A)-   [Non-patent Document 1] Rice Blast and Breeding for its resistance.    Kousaka and Yamazaki eds., p175-186, 1980, Hakuyusha-   [Non-patent Document 2] Wang et al., Plant J 19:55-64, 1999; Bryan    et al. Plant Cell. 12: 2033-46, 2000-   [Non-patent Document 3] Fukuoka and Okuno, Theor Appl Genet.    03:185-190, 2001-   [Non-patent Document 4] Wang et al., Genet 136:1421-1434, 1994

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

The present invention was accomplished under such circumstances. Theproblems to be solved by the present invention are to isolate andidentify genes involved in field resistance to blast by map-basedcloning, and to provide methods for modifying blast field resistance inplants using the genes.

Means for Solving the Problems

The present invention relates to genes controlling blast resistance inplants. The allele pi21 of the Pi21 gene was known to confer rice (Oryzasativa L.) with field resistance to blast and to exist at some positionin the vast region of rice chromosome 4. The present inventors aimed toelucidate the existence region and to isolate the gene as a single gene.

First, the present inventors performed a detailed linkage analysis ofthe pi21 region using a large-scale segregating population indispensablefor map-based cloning, in order to create a genetic map of the pi21region. The inventors obtained a backcross population by continuouslybackcrossing a paddy rice variety, Nipponbare or Aichi Asahi, comprisinga susceptibility allele Pi21 which does not suppress blotch progression,with a Japanese upland rice variety, Owarihatamochi comprising aresistance allele pi21 which suppresses blotch progression. When alinkage analysis with RFLP markers was performed for the obtainedbackcross population, it was confirmed that the pi21 gene locus islocated between the RFLP markers G271 and G317.

Next, using the RFLP markers RA3591 and 13S1, which are located on eachside of the pi21 locus, the present inventors selected organisms withchromosomal recombination near the pi21 locus in order to create a moreaccurate genetic map of the pi21 region. The present inventors alsoselected organisms with chromosomal recombination near the pi21 locus,by searching an F2 population obtained by crossing a line having theresistance allele from Owarihatamochi with a line having the geneticbackground of a Japanese paddy rice variety and the susceptibilityallele from the Indian paddy rice variety Kasalath. As a result ofcreating a detailed linkage map using these organisms and the producedDNA markers, the pi21 gene locus turned out to be located in the genomicregion of about 25 kb sandwiched between the SSCP marker Pa102484 andthe SNP marker P702D3_(—)#12. Further, the nucleotide sequence of PACclone P702D03 that was considered to include the pi21 gene wasdetermined. Moreover, by analyzing the nucleotide sequence of the 25-kbcandidate genomic region in the resistant variety Owarihatamochi and thesusceptibility varieties Aichi Asahi and Kasalath, it was found that thepi21 gene is located in the genomic region of about 1.8 kb sandwichedbetween the SNP markers P702D03_(—)#38 and P702D03_(—)#80.

Thus, the present inventors designed primers that could amplify acorresponding portion using the already obtained nucleotide sequenceinformation of Nipponbare, and then compared the nucleotide sequences ofthe genomic PCR and RT-PCR products between the susceptibility varietiesNipponbare and Aichi Asahi and the resistant variety Owarihatamochi. Asa result, it was found that there are two DNA mutations in the exonregion of the gene in the resistant variety compared to thesusceptibility varieties. It was demonstrated that in contrast to thesusceptibility varieties, the resistant variety has deletions of 7 aminoacids and 16 amino acids, and that these mutations are related to blotchprogression caused by blast infection. That is, the present inventionrelates to the pi21 gene that controls plant resistance to blast, andspecifically provides the following inventions:

-   [1] a DNA of any one of the following (a) to (h):

(a) a DNA that encodes a protein comprising the amino acid sequence ofSEQ ID NO: 3 or 22,

(b) a DNA comprising a coding region of the nucleotide sequence of SEQID NO: 1, 2, 20, or 21,

(c) a DNA encoding a protein which comprises an amino acid sequence witha substitution, deletion, addition, and/or insertion of one or moreamino acids in the amino acid sequence of SEQ ID NO: 3 or 22, and whichhas a function equivalent to that of a protein comprising the amino acidsequence of SEQ ID NO: 3 or 22,

(d) a DNA which hybridizes under stringent conditions to a DNAcomprising the nucleotide sequence of SEQ ID NO: 1, 2, 20, or 21, andwhich encodes a protein having a function equivalent to that of aprotein comprising the amino acid sequence of SEQ ID NO: 3 or 22,

(e) a DNA that encodes a protein comprising the amino acid sequence ofSEQ ID NO: 6,

(f) a DNA comprising a coding region of the nucleotide sequence of SEQID NO: 4 or 5,

(g) a DNA encoding a protein which comprises an amino acid sequence witha substitution, deletion, addition, and/or insertion of one or moreamino acids in the amino acid sequence of SEQ ID NO: 6, and which has afunction equivalent to that of a protein comprising the amino acidsequence of SEQ ID NO: 6, and

(h) a DNA which hybridizes under stringent conditions to a DNAcomprising the nucleotide sequence of SEQ ID NO: 4 or 5, and whichencodes a protein having a function equivalent to that of a proteincomprising the amino acid sequence of SEQ ID NO: 6;

-   [2] a DNA of any one of the following (i) to (iv), having an ability    to confer plants with field resistance to blast:

(i) a DNA that encodes an RNA complementary to a transcription productof the DNA of any one of (a) to (d) in [1],

(ii) a DNA that encodes an RNA having the ribozyme activity tospecifically cleave a transcription product of the DNA of any one of (a)to (d) in [1],

(iii) a DNA that encodes an RNA which inhibits expression of the DNA ofany one of (a) to (d) in [1] by a co-suppression effect, and

(iv) a DNA that encodes an RNA having RNAi activity to specificallycleave a transcription product of the DNA of any one of (a) to (d) in[1];

-   [3] the DNA of [2], wherein the plant is rice, wheat, barley, oat,    corn, Job's tears, Italian ryegrass, perennial ryegrass, timothy,    meadow fescue, millet, foxtail millet, or sugarcane;-   [4] a vector comprising the DNA of any one of [1] to [3];-   [5] a transformed cell that maintains the DNA of any one of [1] to    [3] in an expressible state;-   [6] a transformed plant cell into which the DNA of any one of (a)    to (d) in [1] has been introduced;-   [7] a transformed plant cell into which the DNA of [2] or [3] has    been introduced;-   [8] the transformed plant cell of [6] or [7], wherein the plant is    rice, wheat, barley, oat, corn, Job's tears, Italian ryegrass,    perennial ryegrass, timothy, meadow fescue, millet, foxtail millet,    or sugarcane;-   [9] a transformed plant comprising the transformed cell of any one    of [6] to [8];-   [10] a transformed plant that is a progeny or clone of the    transformed plant of [8];-   [11] a propagation material of the transformed plant of [9] or [10];-   [12] a method for producing the transformed plant of [9] or [10],    which comprises the step of introducing into a plant cell the DNA of    any one of (a) to (d) in [1] or the DNA of [2] or [3], and then    regenerating a plant from the plant cell;-   [13] a method for conferring a plant with field resistance to blast,    which comprises the step of expressing the DNA of [2] or [3] in a    cell of the plant;-   [14] the method of [13], wherein the plant is rice, wheat, barley,    oat, corn, Job's tears, Italian ryegrass, perennial ryegrass,    timothy, meadow fescue, millet, foxtail millet, or sugarcane;-   [15] a protein encoded by the DNA of any one of (a) to (d) in [1];-   [16] a method for producing the protein of [15], which comprises the    step of culturing a transformed cell comprising a vector that    comprises the DNA of any one of (a) to (d) in [1], and then    collecting a recombinant protein from the cell or its culture    supernatant;-   [17] an antibody that binds to the protein of [15];-   [18] a DNA comprising at least 15 consecutive nucleotides    complementary to the DNA of [1] or a complementary sequence thereof;-   [19] an agent that increases field resistance to blast in a plant,    which comprises any one of the DNA of [2] or [3] or the vector    comprising the DNA;-   [20] a primer set that amplifies all or a part of the nucleotide    sequence of SEQ ID NO: 1, 4, or 20;-   [21] a primer set, that is at least any one of the following (a) to    (c):

(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 8, and a DNAcomprising the nucleotide sequence of SEQ ID NO: 9,

(b) a DNA comprising the nucleotide sequence of SEQ ID NO: 16, and a DNAcomprising the nucleotide sequence of SEQ ID NO: 17, and

(c) a DNA comprising the nucleotide sequence of SEQ ID NO: 26, and a DNAcomprising the nucleotide sequence of SEQ ID NO: 27;

-   [22] a DNA comprising the nucleotide sequence of SEQ ID NO: 7, 10,    18, 19, 23, or 25;-   [23] a method comprising the following steps (a) to (c):

(a) preparing a DNA sample from a test plant,

(b) amplifying the DNA region described in [1] from the DNA sample, and

(c) comparing the molecular weight or the nucleotide sequence of theamplified DNA fragment with that of the DNA of (e) or (f) in [1],

which is a method that judges the test plant to have field resistance toblast when the molecular weight or nucleotide sequence is consistentwith that of the DNA of (e) or (f) in [1];

-   [24] a method comprising the following steps (a) to (d):

(a) preparing a DNA sample from a test plant,

(b) amplifying the DNA region described in [1] from the DNA sample,

(c) separating the amplified double-stranded DNA on a non-denaturatinggel, and

(d) comparing the mobility of the separated double-stranded DNA on thegel with that of the DNA of (e) or (f) in [1],

which is a method that judges the test plant to have field resistance toblast when the mobility on the gel is consistent with that of the DNA of(e) or (f) in [1];

-   [25] a method comprising the following steps (a) to (e):

(a) preparing a DNA sample from a test plant,

(b) amplifying the DNA region described in [1] from the DNA sample,

(c) dissociating the amplified DNA into single-stranded DNAs,

(d) separating the dissociated single-stranded DNAs on anon-denaturating gel, and

(e) comparing the mobility of the separated single-stranded DNAs on thegel with that of the DNA of (e) or (f) in [1],

which is a method that judges the test plant to have field resistance toblast when the mobility on the gel is consistent with the DNA of (e) or(f) in [1];

-   [26] a method comprising the following steps (a) to (d):

(a) preparing a DNA sample from a test plant,

(b) amplifying the DNA region described in [1] from the DNA sample,

(c) separating the amplified DNA on a gel with a gradually increasingconcentration of a DNA denaturant, and

(d) comparing the mobility of the separated DNA on the gel, with that ofthe DNA of (e) or (f) in [1],

which is the method that judges the test plant to have field resistanceto blast when the mobility on the gel is consistent with that of the DNAof (e) or (f) in [1];

-   [27] a method for selecting a plant having field resistance to    blast, which comprises the following steps (a) and (b):

(a) producing a hybrid variety by crossing a plant having fieldresistance to blast with a plant having an arbitrary function, and

(b) judging whether the plant produced in step (a) has field resistanceto blast by the method of any one of [23] to [26];

-   [28] a method for judging a test rice plant to have field resistance    to blast when a molecular marker linked to the DNA of [1] shows the    same genotype as that in a rice plant having field resistance to    blast;-   [29] the method of [28], wherein the molecular marker comprises the    DNA of SEQ ID NO: 10;-   [30] a method for selecting a rice plant having field resistance to    blast, wherein the method comprises the following steps (a) and (b):

(a) producing a hybrid variety by crossing a rice plant having fieldresistance to blast with a rice plant having an arbitrary function, and

(b) judging whether the rice plant produced in step (a) has fieldresistance to blast using the method of [28] or [29];

-   [31] a method of screening for an agent that prevents or ameliorates    blast in a plant, wherein the method comprises the following    steps (a) to (c):

(a) contacting a test compound with a transcription product of the DNAof any one of (a) to (d) in [1],

(b) detecting the binding of the transcription product of the DNA of anyone of (a) to (d) in [1] to the test compound, and

(c) selecting a test compound that binds to the transcription product ofthe DNA of any one of (a) to (d) in [1];

-   [32] a method of screening for an agent that prevents or ameliorates    blast in a plant, wherein the method comprises the following    steps (a) to (c):

(a) contacting a test compound with a cell collected from a plant,

(b) measuring the expression level of a transcription product of the DNAof any one of (a) to (d) in [1], and

(c) selecting a test compound that decreases the expression level of thetranscription product as compared to when the test compound is notcontacted;

-   [33] a method of screening for an agent that prevents or ameliorates    blast in a plant, which comprises the following steps (a) to (d):

(a) providing a cell or cell extract comprising a DNA in which areporter gene is operably linked downstream of a promoter region of theDNA of any one of (a) to (d) in [1],

(b) contacting a test compound with the cell or cell extract,

(c) measuring the expression level of the reporter gene in the cell orcell extract, and

(d) selecting a test compound that decreases the expression level of thereporter gene as compared to when the test compound is not contacted;

-   [34] a method of screening for an agent that prevents or ameliorates    blast in a plant, which comprises the following steps (a) to (d):

(a) regenerating a transformed plant from the transformed plant cell of[6],

(b) contacting the blast fungus and a test compound with the transformedplant, and

(c) selecting a test compound that suppresses blast in the transformedplant as compared to when the test compound is not contacted; and

-   [35] a kit for use in the screening method of any one of [31] to    [34].

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows photographs indicating blotches of blast on the lineAA-pi21 having the pi21 gene with the genetic background of Aichi Asahi(left), and those on Aichi Asahi (right).

FIG. 2 shows detailed linkage maps of the pi21 gene region, and analignment map of genomic clones. FIGS. 2A and 2B show the genetic mapscreated using segregating populations of 72 samples and 1014 samples.FIG. 2C shows the alignment map with PAC clones of Nipponbare. FIG. 2Dshows a detailed genetic map of the pi21 gene region, and indicates acandidate genomic region.

FIG. 3 shows a structure of a pi21 candidate gene, and a comparisonbetween genomic nucleotide sequences of Nipponbare and Aichi Asahi andthat of Owarihatamochi.

FIG. 4 shows photographs indicating blotches that appeared on thetransformants of the resistant line AApi21 into which the Pi21 gene fromNipponbare was introduced. A: The vector alone was introduced. B: Onecopy of the Pi21 gene was introduced. C: Three or more copies of thePi21 gene were introduced.

BEST MODE FOR CARRYING OUT THE INVENTION

The pi21 gene, an allele of the susceptibility gene Pi21 which does notsuppress the progression of rice blast, was until now known to belocated somewhere in the vast region of rice chromosome 4, as a genethat confers rice with field resistance to blast. By using the map-basedcloning technique, the present inventors narrowed down the pi21 generegion on rice chromosome 4, and finally succeeded in identifying it asa single gene. Moreover, they also succeeded in isolating the Pi21 gene,an allele of the pi21 gene.

As used herein, the term “blast” means the discoloring or necrotizationof a plant, or part of a plant infected with a blast fungus, or apathological feature (blotch) recognized thereby. The blotches of blastappear in every part of the plant, and blast is called seedling blast,leaf blast, panicle blast, spikelet blast, node blast, and leaf node(ligule) blast and such, according to the part where the blotchesappear. “Blast” in the present invention includes blast occurring on anyof these parts. A “rice blast fungus” which causes blast in rice iscalled Magnaporthe grisea or Magnaporthe oryzae, although there is nounified scientific name at present. Moreover, the blast fungus has ateleomorph name, Magnaporthe oryzae, and a corresponding anamorph name,Pyricularia oryzae, which are used depending on the situation. The blastfungus in the present invention includes all these blast fungi,regardless of their names.

As used herein, the term “blast susceptibility” means the property of aplant to be infected with blast (sometimes means that the symptoms aresignificant). The term “field resistance to blast” means the differencein symptoms or the property of suppressing the number or size ofblotches, which are recognized as the difference in the number or sizeof blotches between varieties or lines (within the same plant species)when plants are infected with the blast fungus. The term “trueresistance” means the property of a plant to cause cell death by ahypersensitive reaction in cells invaded by the blast fungus to preventinfection.

The present invention provides the blast susceptibility gene Pi21,involved in blast of plants, and pi21, a gene conferring fieldresistance to blast.

More specifically, the Pi21 gene of the present invention comprises thefollowing:

(a) a DNA encoding a protein comprising the amino acid sequence of SEQID NO: 3 or 22;

(b) a DNA comprising the coding region of the nucleotide sequence of SEQID NO: 1, 2, 20, or 21;

(c) a DNA encoding a protein which comprises an amino acid sequence witha substitution, deletion, addition, and/or insertion of one or moreamino acids in the amino acid sequence of SEQ ID NO: 3 or 22, and whichhas a function equivalent to that of a protein comprising the amino acidsequence of SEQ ID NO: 3 or 22; and

(d) a DNA which hybridizes with a DNA comprising the nucleotide sequenceof SEQ ID NO: 1, 2, 20, or 21 under a stringent condition, and whichencodes a protein having the function equivalent to a protein comprisingthe amino acid sequence of SEQ ID NO: 3 or 22.

Furthermore, the pi21 gene of the present invention specificallycomprises the following:

(a) a DNA encoding a protein comprising the amino acid sequence of SEQID NO: 6;

(b) a DNA comprising the coding region of the nucleotide sequence of SEQID NO: 4 or 5;

(c) a DNA encoding a protein which comprises an amino acid sequence witha substitution, deletion, addition, and/or insertion of one or moreamino acids in the amino acid sequence of SEQ ID NO: 6, and which hasthe function equivalent to that of a protein comprising the amino acidsequence of SEQ ID NO: 6; and

(d) a DNA which hybridizes with a DNA comprising the nucleotide sequenceof SEQ ID NO: 4 or 5 under a stringent condition, and which encodes aprotein having the function equivalent to a protein comprising the aminoacid sequence of SEQ ID NO: 6.

By using the Pi21 gene or the pi21 gene of the present invention, itbecomes possible, for example, to prepare recombinant proteins orgenerate transformed plants with modified field resistance to blast.

In the present invention, plants from which the genes of the presentinvention are derived include, but are not particularly limited to, forexample, monocotyledons such as rice, corn, wheat, barley, oat, Job'stears, Italian ryegrass, perennial ryegrass, timothy, meadow fescue,millet, foxtail millet, sugarcane, and pearl millet; and dicotyledonssuch as rapeseed, soybean, cotton, tomato, and potato. They also includeflowering plants such as chrysanthemum, rose, carnation, and cyclamen,but are not particularly limited thereto.

There is no particular restriction on the forms of the “Pi21 gene” andthe “pi21 gene” of the present invention, as long as they can encode the“Pi21 protein” and “pi21 protein”, respectively; and the “Pi21 gene” andthe “pi21 gene” each comprises a genomic DNA, chemically synthesized DNAand so on as well as a cDNA. Moreover, the Pi21 gene and the pi21 genecomprise a DNA with any nucleotide sequence based on genetic codedegeneracy, as long as they encode the Pi21 protein and the pi21protein, respectively.

One skilled in the art can prepare genomic DNAs and cDNAs by usingconventional means. Genomic DNAs can be prepared, for example, byextracting genomic DNAs from a plant; constructing a genomic library (aplasmid, phage, cosmid, BAC, PAC or the like can be used as a vector);developing it; and performing colony hybridization or plaquehybridization using a probe prepared based on the Pi21 gene or the pi21gene (for example, the DNA of any one of SEQ ID NO: 1, 2, 4, 5, 20, or21). Alternatively, genomic DNAs can be prepared by preparing primersspecific for the Pi21 gene or the pi21 gene and performing PCR by usingthese primers. cDNAs can be prepared, for example, by synthesizing cDNAsbased on mRNAs extracted from a plant; inserting them into vectors suchas XZAP to create a cDNA library; developing it; and performing colonyhybridization or plaque hybridization as described above. They can alsobe prepared by performing PCR.

Further, since the Pi21 gene or the pi21 gene is considered to be widelypresent in the plant kingdom, the Pi21 gene or the pi21 gene alsoincludes not only genes in rice but also homologous genes present invarious plants. Herein, the term “homologous gene” refers to a gene invarious plants that encodes a protein having a physiological function(for example, blast susceptibility or field resistance to blast) similarto that of the Pi21 gene product or the pi21 gene product in rice.

Methods for isolating homologous genes well known to one skilled in theart include the hybridization technique (Southern E. M., Journal ofMolecular Biology, Vol. 98, 503, 1975) and the polymerase chain reaction(PCR) technique (Saiki, R. K., et al. Science, vol. 230, 1350-1354,1985; Saiki, R. K. et al. Science, vol. 239, 487-491, 1988).Specifically, one skilled in the art can usually isolate homologousgenes of the Pi21 gene or the pi21 gene from various plants, by using asa probe the nucleotide sequences (for example, the sequence of any oneof SEQ ID NO: 1, 2, 4, 5, 20, or 21) of the rice Pi21 gene or pi21 gene,or a part of it, or by using as primers oligonucleotides whichspecifically hybridize to the Pi21 gene or the pi21 gene.

In order to isolate DNAs encoding such homologous genes, thehybridization reaction is usually performed under stringent conditions.Examples of stringent hybridization conditions include the conditions of6 M urea, 0.4% SDS, and 0.5×SSC, or hybridization conditions ofequivalent stringency. Isolation of DNAs with higher homology can beexpected by using conditions with higher stringency, for example, 6 Murea, 0.4% SDS, and 0.1×SSC. The sequences of the isolated DNAs can bedetermined by a known method. Homology of isolated DNAs indicates asequence identity of at least 50% or more, more preferably 70% or more,still more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99%or more) over the entire amino acid sequence. Sequence homology can bedetermined using the programs of BLASTN (nucleic acid level) or BLASTX(amino acid level) (Altschul et al. J. Mol. Biol. 215: 403-410, 1990).The programs are based on the algorithm BLAST by Karlin and Altschul(Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990; Proc. Natl. Acad. Sci.USA, 90: 5873-5877, 1993). When analyzing a nucleotide sequence byBLASTN, parameters are set to, for example, score=100 and wordlength=12.When analyzing an amino acid sequence by BLASTX, parameters are, forexample, set to score=50 and wordlength=3. Alternatively, an amino acidsequence can be analyzed using Gapped BLAST program as indicated byAltschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When BLAST andGapped BLAST programs are used, the default parameters of each programare used. The specific procedures of these analysis methods are known.

The present invention also provides the following DNAs that are used tosuppress the plant endogenous Pi21 gene expression:

(a) a DNA encoding an RNA complementary to a transcription product ofthe Pi21 gene,

(b) a DNA encoding an RNA that has the ribozyme activity to specificallycleave a transcription product of the Pi21 gene,

(c) a DNA encoding an RNA which inhibits Pi21 gene expression by aco-suppression effect, and

(d) a DNA encoding an RNA that has the RNAi activity to specifically cuta transcription product of the Pi21 gene.

These DNAs can suppress blotch progression by blast in plants.

In the present invention, plants in which the Pi21 gene expression issuppressed are not particularly limited, and any plant desired to beconferred field resistance to blast can be used; however, agriculturalcrops and ornamental plants are suitable from an industrial viewpoint.Useful agricultural crops include, but are not particularly limited to,monocotyledons such as rice, corn, wheat, barley, oats, Job's tears,Italian ryegrass, perennial ryegrass, timothy, meadow fescue, millet,foxtail millet, sugarcane, and pearl millet; and dicotyledons such asrapeseed, soybean, cotton, tomato, and potato. Ornamental plants includeflowering plants such as chrysanthemum, rose, carnation, and cyclamen,but are not limited thereto. Plants susceptible to the rice blast fungusinclude pasture grasses such as barley, Italian ryegrass, and meadowfescue; and corn. In addition, many plants have been reported as thosewhich the blast fungus separated from rice can parasitize, includingtribe Oryzeae such as Leersia oryzoides and wild rice; tribe Poeae;tribe Triticeae; tribe Aveneae such as oat; tribe Chloridinae such asEragrostis curvula; and tribe Paniceae such as foxtail millet andcrabgrass. These plants are also included in the plants on which fieldresistance to blast can be conferred.

As used herein, “suppression of Pi21 gene expression” includessuppression of gene transcription and suppression of translation to aprotein. Moreover, it includes not only the complete arrest of DNAexpression, but also reduction of expression.

One embodiment of “a DNA used to suppress Pi21 gene expression” is a DNAwhich encodes an antisense RNA complementary to the Pi21 gene. Using thetemporal gene expression method, the antisense effect in a plant cellwas demonstrated for the first time through the fact that an antisenseRNA introduced by electroporation exhibited an antisense effect in aplant (Ecker and Davis, Proc. Natl. Acad. USA, 83: 5372, 1986).Thereafter, expression of antisense RNAs in tobacco and petunia havealso been reported to reduce target gene expression (Krol et. al.,Nature 333: 866, 1988). At present, it is established as a means tosuppress gene expression in plants.

There are a number of factors involved in the action of antisensenucleic acids in suppressing target gene expression, as indicated asfollows: inhibiting transcription initiation by forming triple strands;suppressing transcription by hybridizing with a site where RNApolymerase has formed a local open loop structure; inhibitingtranscription by hybridizing with the RNA being synthesized; suppressingsplicing by hybridizing with an intron-exon junction; suppressingsplicing by hybridizing with the site of spliceosome formation;suppressing transfer from the nucleus to the cytoplasm by hybridizingwith an mRNA; suppressing splicing by hybridizing with a poly(A)addition site or capping site; suppressing translation initiation byhybridizing with a translation initiation factor binding site;suppressing translation by hybridizing with a ribosome binding site nearthe initiation codon; preventing peptide chain elongation by hybridizingwith an mRNA translation region or polysome binding site; andsuppressing gene expression by hybridizing with a nucleic acid-proteininteraction site. Antisense nucleic acids suppress target geneexpression by inhibiting transcription, splicing, or translation process(Hirashima and Inoue, 1993, “Shin Seikagaku Jikken Kouza (NewBiochemistry Experimentation Lectures) 2, Kakusan (Nucleic Acids) IV,Idenshi No Fukusei To Hatsugen (Replication and Expression of Genes)”,The Japanese Biochemical Society Ed., Tokyo Kagaku Dojin, pp. 319-347).

The antisense sequences used in the present invention can suppress theexpression of a target gene by any of the above actions. As oneembodiment, an antisense sequence designed to be complementary to anuntranslated region close to the 5′ end of the mRNA of a gene will beeffective in inhibiting translation of that gene. However, a sequencecomplementary to a coding region, or to a 3′-end untranslated region canalso be used. In this way, DNAs comprising antisense sequences of agene's translated regions as well untranslated regions are included inthe antisense DNAs that can be used in the present invention. Anantisense DNA to be used herein is ligated downstream of an appropriatepromoter, and a sequence comprising a transcription termination signalis preferably ligated to the 3′ side of the DNA.

Antisense DNAs can be prepared, for example, based on the DNA sequenceof SEQ ID NO: 1, 2, 20, or 21 by using the phosphorothioate method(Stein, Nucleic Acids Res., 16: 3209-3221, 1988) and such. DNAs thusprepared can be transformed into a desired plant using known methods.Antisense DNA sequences are preferably sequences complementary to atranscription product of an endogenous gene of the plant to betransformed, but need not be perfectly complementary as long as they caneffectively inhibit gene expression. The transcribed RNAs are preferably90% or more (for example, 95%, 96%, 97%, 98%, 99% or more) complementaryto the transcription products of the target genes. In order toeffectively inhibit target gene expression using an antisense sequence,an antisense DNA should comprise at least 15 nucleotides or more,preferably 100 nucleotides or more, and even more preferably 500nucleotides or more. Antisense DNAs to be used are generally less than 5kb, and preferably less than 2.5 kb long.

Suppression of the endogenous Pi21 gene expression can also be carriedout using DNAs encoding ribozymes. The term “ribozyme” refers to an RNAmolecule having catalytic activity. Some ribozymes have many differentactivities. Among them, research on ribozymes as RNA-cleaving enzymeshas enabled designing ribozymes to cleave RNAs at specific sites.Ribozymes include those of 400 nucleotides or more, such as M1RNA inRNaseP, or the group 1 intron type ribozymes. In contrast, there arealso hammerhead-type or hairpin-type ribozymes that comprise an activedomain of about 40 nucleotides (Koizumi, M. and Ohtsuka, E., 1990,Protein, Nucleic acid and Enzyme, 35: 2191-2200).

For example, the self-cleaving domain of a hammerhead type ribozymecleaves at the 3′ side of C15 in G13U14C15. Base pairing between U14 andA9 is important for ribozyme activity. It has been shown that cleavagecan occur if A or U instead of C is at the 15th position (Koizumi, M. etal., 1988, FEBS Lett. 228: 228-230). If the substrate-binding site ofthe ribozyme is designed to be complementary to the RNA sequencesadjacent to the target site, a restriction enzyme-like RNA-cleavingribozyme can be created that recognizes the sequence UC, UU, or UAwithin the target RNA (Koizumi et al., 1988, FEBS Lett. 239: 285;Koizumi, M. and Ohtsuka, E., 1990, Protein, Nucleic acid and Enzyme, 35:2191; Koizumi et al., 1989, Nucleic Acids Res. 17: 7059).

Hairpin type ribozymes are also useful for objectives of the presentinvention. A hairpin type ribozyme can be found, for example, in theminus strand of tobacco ringspot virus satellite RNA (Buzayan, Nature323: 349, 1986). It has also been shown that this ribozyme can bedesigned to target-specifically cleave an RNA (Kikuchi and Sasaki,Nucleic Acids Res. 19: 6751, 1992; Kikuchi, H., Kagaku to Seibutsu(Chemistry and Biology) 30: 112, 1992).

In order to be transcribed in plant cells, a ribozyme designed to cleavea target is linked to a transcription termination sequence or a promotersuch as the cauliflower mosaic virus 35S promoter. However, if extrasequences are added to the 5′- or the 3′-end of the transcribed RNA, theribozyme activity can be lost. In this case, another cis-acting trimmingribozyme can be placed in the 5′ or 3′ side of the ribozyme portion toprecisely trim only the ribozyme portion from the transcribed RNAcomprising the ribozyme (Taira et al., Protein Eng. 3: 733, 1990;Dzianott and Bujarski, Proc. Natl. Acad. Sci. USA 86: 4823, 1989;Grosshans and Cech, Nucleic Acids Res. 19: 3875, 1991; Taira et al.,Nucleic Acid Res. 19: 5125, 1991).

In addition, these structural units can be arranged in tandem to cleavemultiple sites within a target gene, thus achieving greater effects(Yuyama et al., Biochem. Biophys. Res.

Commun. 186: 1271, 1992). By using these kinds of ribozymes, thetranscription products of the target genes of the present invention canbe specifically cleaved, and the gene expression can be suppressed.

Suppression of endogenous gene expression can also be achieved by“co-suppression” resulting from transformation with a DNA comprising asequence identical or similar to a target gene sequence. The term“co-suppression” refers to the phenomenon in which, when a genecomprising a sequence identical or similar to that of the targetendogenous gene is introduced into plants by transformation, expressionof both the introduced exogenous gene and the target endogenous gene issuppressed. The detailed mechanism of co-suppression is unknown, but itis frequently observed in plants (Curr. Biol., 7: R793, 1997; Curr.Biol. 6: 810, 1996).

For example, to obtain a plant in which the Pi21 gene is co-suppressed,plants of interest are transformed with a vector DNA constructed toexpress the Pi21 gene or a DNA comprising a similar sequence, and plantswith a characteristic of a mutant Pi21, i.e., plants with fieldresistance to blast, are selected from the plants thus obtained. Genesto be used for co-suppression do not have to be completely identical tothe target gene; however, they have sequence identity of at least 70% ormore, preferably 80% or more, and more preferably 90% or more (forexample, 95%, 96%, 97%, 98%, 99% or more).

In addition, suppression of endogenous gene expression in the presentinvention can also be achieved by transforming a plant with a genecomprising a characteristic that is dominant-negative to the targetgene. A “gene comprising a dominant-negative characteristic” refers to agene that, when expressed, has the function of eliminating or reducingthe activity of an original endogenous wild-type gene of the plant.

Another embodiment of “a DNA used to suppress Pi21 gene expression” is aDNA which encodes a double-stranded RNA (dsRNA) complementary to atranscription product of an endogenous Pi21 gene. By introducing a dsRNAcomprising a sequence identical or similar to a target gene sequenceinto a cell, a phenomenon called RNAi (RNA interference) can be caused,where expression of both the introduced foreign gene and the targetendogenous gene are suppressed. When a dsRNA of about 40 to severalhundred base pairs is introduced into a cell, an RNase III-like nucleasecomprising a helicase domain, called Dicer, cuts out about 21 to 23 basepair portions from the 3′-terminus of the dsRNA in the presence of ATP,thereby producing an siRNA (short interference RNA). This siRNA binds toa specific protein to form a nuclease complex (RISC: RNA-inducedsilencing complex). This complex recognizes and binds to a sequenceidentical to the siRNA, and cuts the transcription product (mRNA) of thetarget gene at the central part of the siRNA with the RNaseIII-likeenzymatic activity. Apart from this pathway, an antisense strand ofsiRNA binds to an mRNA to act as a primer of an RNA-dependent RNApolymerase (RsRP), thereby synthesizing a dsRNA. Another pathway is alsoconsidered in which this dsRNA serves again as a substrate of Dicer,produces a new siRNA, and amplifies its effect.

RNAi was first discovered in nematodes (Fire, A. et al., Potent andspecific genetic interference by double-stranded RNA in Caenorhabditiselegans. Nature 391, 806-811, 1998). At present, it is observed not onlyin nematodes, but also in various organisms such as plants,Nemathelminthes, Drosophila, and protozoa (Fire, A. RNA-triggered genesilencing. Trends Genet. 15, 358-363 (1999); Sharp, P. A. RNAinterference 2001. Genes Dev. 15, 485-490 (2001); Hammond, S. M., Caudy,A. A. & Hannon, G. J. Post-transcriptional gene silencing bydouble-stranded RNA. Nature Rev. Genet. 2, 110-119 (2001); Zamore, P. D.RNA interference: listening to the sound of silence. Nat Struct Biol. 8,746-750 (2001)). In these organisms, it was confirmed that target geneexpression was actually suppressed by externally introducing a dsRNA.Further, RNAi is now being used as a method for creating knockoutorganisms.

When RNAi was initially found, only a dsRNA of a certain length (40bases) or more was thought to be effective. However, Tuschl et al. ofRockefeller University, United States, reported that by introducing ashort dsRNA (siRNA) of about 21 base pairs into a cell, an RNAi effectwas obtained in a mammalian cell without causing an antiviral reactionby PKR (Tuschl, Nature, 411, 494-498 (2001)). Thus, RNAi has suddenlyattracted attention as a technique applicable to differentiatedmammalian cells such as human cells.

The DNAs of the present invention comprise an antisense code DNAencoding an antisense RNA corresponding to any region of a transcriptionproduct (mRNA) of a target gene, and a sense code DNA encoding a senseRNA corresponding to any region of the mRNA. The above-mentionedantisense RNA and sense RNA can be expressed from the above-mentionedantisense code DNA and sense code DNA. A dsRNA can also be produced fromthese antisense RNA and sense RNA. A target sequence in the presentinvention is not particularly limited, as long as the Pi21 geneexpression is suppressed by introducing into a cell a dsRNA comprising asequence identical or similar to the target sequence. An example of thetarget sequence includes a sequence of 3′-untranslated region of thePi21 gene. A sequence of 3′-untranslated region of the Pi21 gene isshown in SEQ ID NOs: 11 and 24.

An expression system of dsRNAs of the present invention is maintained asfollows in a vector or the like: an antisense RNA and a sense RNA areexpressed from the same vector; or an antisense RNA and a sense RNA areexpressed from different vectors, respectively. For example, whenexpressing an antisense RNA and a sense RNA from the same vector, anantisense RNA expression cassette and sense RNA expression cassette areeach constructed, in which a promoter like the pol III system that canexpress a short RNA is connected upstream of the antisense code DNA andsense code DNA, respectively, and these cassettes are then inserted intoa vector in the same direction or in the opposite direction.

An expression system can also be constructed in which an antisense codeDNA and a sense code DNA are arranged in opposite directions ondifferent strands so that they face each other. This construct can carryone double-stranded DNA (siRNA code DNA) in which an antisenseRNA-encoding strand and a sense RNA-encoding strand are paired, andpromoters which are oppositely oriented on both sides so that theantisense RNA and the sense RNA can be expressed from each strand. Inthis case, in order to prevent addition of an excess sequence downstreamof the sense RNA and the antisense RNA, a terminator is preferablyplaced at the 3′-terminus of each strand (the antisense RNA-encodingstrand and the sense RNA-encoding strand). A sequence of four or moreconsecutive A (adenine) bases can be used for this terminator. Moreover,in this palindrome type expression system, the kinds of two promotersare preferably different to each other.

When expressing an antisense RNA and sense RNA from different vectors,for example, the following procedures are performed: An antisense RNAexpression cassette and a sense RNA expression cassette are constructed,in each of which a promoter such as the pol III system that can expressa short RNA, is connected upstream of the antisense code DNA or thesense code DNA; and then these cassettes are maintained in differentvectors.

As for RNAi, an siRNA may be used as a dsRNA. The term “siRNA” means adouble-stranded RNA including short strands that exhibit no toxicitywithin a cell, and is not limited to the full length of 21 to 23 basepairs reported by Tuschl et al. (ibid.); and is not particularlylimited, as long as the length is in such a range that it exhibits notoxicity. For example, an siRNA can be 15 to 49 base pairs, preferably15 to 35 base pairs, and still more preferably 21 to 30 base pairs inlength. Alternatively, length of the final double-stranded RNA portionthat results from transcription of an siRNA to be expressed, can be 15to 49 base pairs, preferably 15 to 35 base pairs, and more preferably 21to 30 base pairs, for example.

As a DNA of the present invention, such a construct that is produced byinserting a suitable sequence (an intron sequence is preferable) betweenthe inverted repeats of a target sequence (Smith, N. A., et al. Nature,407: 319, 2000; Wesley, S. V. et al. Plant J. 27: 581, 2001; Piccin, A.et al. Nucleic Acids Res. 29: E55, 2001) and yields a double-strandedRNA having a hairpin structure (self-complementary ‘hairpin’ RNA(hpRNA)), can also be used.

Although a DNA used for RNAi is not required to be completely the sameas a target gene, it has a sequence identity of at least 70% or more,preferably 80% or more, still more preferably 90% or more (for example,95%, 96%, 97%, 98%, 99% or more). The sequence identity can bedetermined by using the above-mentioned procedures.

The double-stranded RNA portions in dsRNAs, in which RNAs are paired,are not necessarily completely paired, but may comprise unpairedportions due to a mismatch (corresponding bases are not complementary),a bulge (there is no corresponding base on one strand) or the like. Inthe present invention, both bulges and mismatches may be included indouble-stranded RNA regions where RNAs are paired with each other indsRNAs.

The present invention also provides vectors and transformed cellscomprising any one of the Pi21 gene, the pi21 gene, and DNAs thatsuppress Pi21 gene expression.

With regard to the above vectors, for example, when the host is E. coli,as long as the vector has an “ori” for amplification in E. coli, suchthat vectors are amplified and prepared in large quantities in E. coli(for example, JM109, DH5α, HB101, and XL1Blue) or such, and further hasa selection gene for transformed E. coli (for example, a drug resistancegene that allows discrimination using a certain drug (ampicillin,tetracycline, kanamycin, or chloramphenicol)), the vectors are notparticularly limited. The vectors include, for example, M13 vectors, pUCvectors, pBR322, pBluescript, and pCR-Script. In addition to the abovevectors, for example, pGEM-T, pDIRECT, and pT7 can also be used for thesubcloning and excision of cDNAs. When using vectors to produce the Pi21gene, the pi21 gene, and the DNAs that suppress Pi21 gene expression,expression vectors are particularly useful. When an expression vector isexpressed in E. coli, for example, it should have the abovecharacteristics in order to be amplified in E. coli. Additionally, whenE. coli such as JM109, DH5α, HB101, or XL1-Blue are used as the host,the vector must have a promoter that allows efficient expression in E.coli, for example, a lacZ promoter (Ward et al. Nature 341:544-546,1989; FASEB J. 6: 2422-2427, 1992), araB promoter (Better et al. Science240:1041-1043, 1988), or T7 promoter. Other examples of the vectorsinclude pGEX-5X-1 (Pharmacia), “QlAexpress system” (QIAGEN), pEGFP, andpET.

Furthermore, the vector may comprise a signal sequence for polypeptidesecretion. When producing polypeptides into the periplasm of E. coli,the pelB signal sequence (Lei, S. P. et al. J. Bacteriol. 169:4379(1987)) may be used as a signal sequence for polypeptide secretion. Forexample, calcium chloride methods or electroporation methods may be usedto introduce the vector into a host cell.

In addition to E. coli, expression vectors derived from mammals (e.g.,pcDNA3 (Invitrogen), pEGF-BOS (Nucleic Acids Res. 18(17): 5322 (1990)),pEF, and pCDM8), insect cells (e.g., “Bac-to-BAC baculovirus expressionsystem” (GIBCO-BRL) and pBacPAK8), plants (e.g., pMH1 and pMH2), animalviruses (e.g., pHSV, pMV, and pAdexLcw), retroviruses (e.g., pZIPneo),yeasts (e.g., “Pichia Expression Kit” (Invitrogen), pNV11 and SP-Q01),and Bacillus subtilis (e.g., pPL608 and pKTHSO) may also be used asvectors for producing the Pi21 gene, the pi21 gene, and the DNAs whichsuppress Pi21 gene expression.

For expression in animal cells such as CHO, COS, and NIH3T3 cells, thevector must have a promoter necessary for expression in such cells, forexample, an SV40 promoter (Mulligan et al. Nature 277: 108 (1979)),MMLV-LTR promoter, EF1α promoter (Mizushima et al. Nucleic Acids Res.18: 5322 (1990)), or CMV promoter. It is even more preferable that thevector comprises a gene for selecting transformants (for example, adrug-resistance gene enabling discrimination by a drug (such as neomycinand G418)). Examples of vectors with such characteristics include pMAM,pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

Introduction of a DNA of the present invention into a cell can becarried out by a method known to one skilled in the art, for example, byan electroporation method.

Further, the present invention provides transformed plant cells intowhich the DNA encoding the Pi21 protein or a DNA suppressing the Pi21gene expression has been introduced; transformed plants derived from thecells; transformed plants which are progenies or clones of thetransformed plants; and propagation materials of the transformed plants.Methods for producing the above-mentioned transformants and propagationmaterials are also provided.

The DNA encoding the Pi21 protein or DNAs suppressing Pi21 geneexpression can be introduced into plant cells by the above methods.

In addition, regeneration of plants is also possible using methods knownto those skilled in the art, according to the type of plant cell (Tokiet al., Plant Physiol., 100: 1503-1507, 1995). In rice, for example, anumber of techniques for producing transformed plants are alreadyestablished, and are widely used in the technical field of the presentinvention. These methods include the method for introducing genes intoprotoplasts using polyethylene glycol and then regenerating plants(suitable for Indian varieties of rice) (Datta et al., In Gene TransferTo Plants. Potrykus, I. and Spangenberg, G. Eds., pp. 66-74, 1995), themethod for introducing genes into protoplasts using electric pulse andthen regenerating plants (suitable for Japanese varieties of rice) (Tokiet al., Plant Physiol. 100: 1503-1507, 1992), the method for directlyintroducing genes into cells using the particle gun method and thenregenerating plants (Christou et al., Bio/technology, 9: 957-962, 1991),and the method for introducing genes via an Agrobacterium, and thenregenerating plants (Hiei et al., Plant J., 6: 271-282, 1994). Thesemethods can be appropriately used in the present invention.

When using the above Agrobacterium method, the method of Nagel et al.(Microbiol. Lett. 67: 325, 1990) is used, for example. In this method, arecombinant vector is transformed into an Agrobacterium, andsubsequently the transformed Agrobacterium is introduced into a cell bya known method such as the leaf disk method. The above-mentioned vectorcomprises an expression promoter so that, for example, the DNA encodingthe Pi21 protein of the present invention or a DNA suppressing the Pi21gene expression is expressed in a plant after introduction into theplant. Generally, DNA encoding the Pi21 protein of the present inventionor a DNA suppressing the Pi21 gene expression is located downstream ofthe promoter, and a terminator is located further downstream of such aDNA. The recombinant vector used for this purpose is suitably selectedby one skilled in the art, depending on the type of plant or method ofintroduction. The above-mentioned promoters include, for example, theCaMV35S derived from cauliflower mosaic virus and the ubiquitin promoterfrom corn (JP-A H2-79983).

Examples of the above-mentioned terminator can be a terminator derivedfrom cauliflower mosaic virus and the terminator from the nopalinesynthase gene; however, the promoter and terminator are not limitedthereto, as long as they function in a plant.

The plants into which the DNA encoding the Pi21 protein of the presentinvention or a DNA suppressing the Pi21 gene expression is introduced,may be explants, or the DNA may be introduced into the cultured cellsprepared from these plants. “Plant cells” in the present inventioninclude, for example, plant cells of a leaf, root, stem, flower, andscutellum in a seed; calluses; and suspension-cultured cells.

In order to efficiently select the cells transformed by introducing theDNA encoding the Pi21 protein of the present invention or a DNAsuppressing the Pi21 gene expression, the above-mentioned recombinantvector is introduced into the plant cells, preferably together with asuitable selection marker gene or a plasmid vector comprising aselection marker gene. The selection marker genes used for this purposeinclude, for example, the hygromycin phosphotransferase gene resistantto the antibiotic hygromycin, the neomycin phosphotransferase resistantto kanamycin or gentamycin, and the acetyltransferase gene resistant tothe herbicide phosphinothricin.

The cells into which the recombinant vector has been introduced areplaced on a known selection medium containing a suitable selection agentdepending on the type of introduced selection marker gene, and thencultured. In this way, the transformed plant cultured cells can beobtained.

Next, plant bodies reproduced from the transformed cells are cultured inan acclimation medium. The acclimated, regenerated plant bodies are thengrown under usual culture conditions to obtain plant bodies having fieldresistance to blast, from which seeds can be obtained once they matureand bear fruit.

The presence of the introduced foreign DNAs in the transformed plantsthat are regenerated and grown in this manner can be confirmed by theknown PCR method or Southern hybridization method, or by analyzing thenucleotide sequences of the DNAs in plant bodies.

In this case, extraction of the DNAs from the transformed plants can becarried out according to the known method by J. Sambrook et al.(Molecular Cloning, the 2nd edition, Cold Spring Harbor LaboratoryPress, 1989).

When analyzing the foreign genes which are present in the regeneratedplant bodies and include the DNAs of the present invention, using thePCR method, an amplification reaction is carried out using as a templatethe DNAs extracted from the regenerated plant bodies as mentioned above.An amplification reaction can also be performed in a reaction mixturecontaining as primers synthesized oligonucleotides which comprisenucleotide sequences suitably selected according to the nucleotidesequences of the DNAs of the present invention or the DNAs modifiedaccording to the present invention. In the amplification reaction,denaturation, annealing, and extension reactions of DNAs can be repeatedseveral tens of times to obtain amplified products of DNA fragmentscomprising the DNA sequences of the present invention. By subjecting thereaction mixture comprising the amplified products, for example, toagarose electrophoresis, the various kinds of amplified DNA fragmentsare fractionated, thereby enabling confirmation of whether a certain DNAfragment corresponds to a DNA of the present invention.

After obtaining a transformed plant in which a DNA encoding the Pi21protein of the present invention or a DNA suppressing the Pi21 geneexpression has been introduced into the chromosome, progenies can beobtained by sexual or asexual reproduction from the plant. Further,propagation materials (for example, seeds, fruits, panicles, tubers,tuberous roots, stocks, calluses, and protoplasts) can also be obtainedfrom the plant or its progenies or clones, and these materials can beused to mass-produce the plants. The present invention comprises plantcells into which the DNA encoding the Pi21 protein or a DNA suppressingthe Pi21 gene expression has been introduced; plants comprising thecells; progenies and clones of the plants; and propagation materials ofthe plants and their progenies and clones. Such plant cells, plantscomprising the cells, progenies and clones of the plants, andpropagation materials of the plants and their progenies and clones, canbe used in methods for conferring plants with field resistance to blast.

The present invention further provides methods for conferring plantswith field resistance to blast, comprising the step of expressing a DNAwhich suppresses Pi21 gene expression in cells of plant bodies. Fieldresistance to blast can be conferred on plants by introducing into plantcells vectors which comprise, in an expressible state, a DNA suppressingPi21 gene expression, using the above-mentioned methods, and byregenerating plants using these cells.

As used herein, the term “'conferring' field resistance to blast” meansnot only to provide a blast field resistance capacity to plants havingno blast field resistance capacity, but also to further increase blastfield resistance capacity of plants which already have it. In thepresent invention, plants in which Pi21 gene expression is suppressedand on which field resistance to blast is conferred, are notparticularly limited; and such plants include, for example, theabove-mentioned plants.

The present invention also provides proteins encoded by the Pi21 gene ofthe present invention, methods for producing the proteins, andantibodies which bind to the proteins.

Recombinant proteins are typically prepared by inserting DNAs encodingproteins of the present invention into appropriate expression vectors,introducing the vectors into appropriate cells, culturing thetransformed cells, and purifying the expressed proteins. Recombinantproteins can be expressed as fusion proteins with other proteins to makepurification easier, for example, as fusion proteins withmaltose-binding protein using Escherichia coli as a host (New EnglandBiolabs, USA, vector pMAL series), as fusion proteins with glutathioneS-transferase (GST) (Amersham Pharmacia Biotech, vector pGEX series), ortagged with histidine (Novagen, pET series). The host cells are notparticularly limited, so long as the cell is suitable for expressing therecombinant proteins. It is possible to use, for example, yeast, variousplant or animal cells, insect cells or such in addition to theabove-described E. coli. Vectors can be introduced into host cells by avariety of methods known to those skilled in the art. For example,introduction methods using calcium ions can be used for introductioninto E. coli (Mandel, M. & Higa, A. Journal of Molecular Biology, Vol.53, 158-162 (1970); Hanahan, D. Journal of Molecular Biology, Vol. 166,557-580 (1983)). Recombinant proteins expressed in the host cells can bepurified and recovered from the host cells or the culture supernatantthereof by known methods in the art. When recombinant proteins areexpressed as fusion proteins with the aforementioned maltose-bindingprotein or such, affinity purification can be carried out easily.

The obtained recombinant proteins can be used to prepare antibodieswhich bind to the proteins. Polyclonal antibodies can be obtained asfollows, for example: Small animals such as rabbits are immunized withthe Pi21 protein a recombinant protein expressed as a fusion proteinwith GST in microorganisms such as E. coli., or its partial peptide toobtain sera. The antibodies are prepared by purifying the sera using,for example, ammonium sulfate precipitation, a protein A or protein Gcolumn, DEAE ion exchange chromatography, or an affinity column coupledwith the Pi21 protein or a synthetic peptide. Monoclonal antibodies canbe prepared as follows: small animals such as mice are immunized withthe Pi21 protein or its partial peptide; the spleen is harvested fromthe mice and ground to separate cells; the cells and mouse myeloma cellsare fused using a reagent such as polyethylene glycol; and from amongthe fused cells (hybridomas) thus obtained, clones which produceantibodies binding to the Pi21 protein are selected. Subsequently, theobtained hybridomas are transplanted into the abdominal cavity of mice,ascites are collected from the mice to prepare monoclonal antibodies,for example, by purifying using ammonium sulfate precipitation, aprotein A or protein G column, DEAE ion exchange chromatography, anaffinity column coupled with the Pi21 protein or a synthetic peptide.The antibodies thus obtained can be used for purification, detection andthe like of the proteins of the present invention. The present inventioncomprises antibodies which bind to the proteins of the presentinvention.

The present invention provides oligonucleotides comprising at least 15nucleotides which are complementary to the pi21 gene, a DNA comprisingthe Pi21 gene, or the complementary strand thereof.

A “complementary strand” herein refers to one strand relative to theother strand in a double-stranded nucleic acid comprising base pairs ofA:T (U for RNA) and G:C. The term “complementary” means not only that asequence is completely complementary in a region of at least 15consecutive nucleotides, but also that a sequence has a homology of atleast 70%, preferably at least 80%, more preferably 90%, still morepreferably 95% or more in the nucleotide sequence. Any algorithm knownto one skilled in the art may be used for determining homology.

The oligonucleotides of the present invention can be used as probes orprimers for detection and amplification of DNAs comprising thenucleotide sequence of SEQ ID NO: 1, 2, 4, 5, 20, or 21. Moreover, theoligonucleotides of the present invention can be used in the form of aDNA array substrate.

When such an oligonucleotide is used as a primer, the length is usually15 by to 100 bp, and preferably 17 by to 30 bp. The primer is notparticularly limited, as long as it can amplify at least a portion of aDNA of the present invention or its complementary strand. When used as aprimer, its 3′ side region is made to be complementary, and arestriction enzyme recognition sequence, a tag or the like can be addedto its 5′ side.

When the above-mentioned oligonucleotide is used as a probe, anyoligonucleotide may be used without particular limitation, as long as itcan specifically hybridize at least a portion of a DNA comprising thenucleotide sequence of SEQ ID NO: 1, 2, 4, 5, 20, or 21 or itscomplementary strand. The probe may be a synthetic oligonucleotide, andusually has a length of at least 15 bp or more.

When an oligonucleotide of the present invention is used as a probe, itis preferably labeled as appropriate. Labeling methods include thefollowing, for example: a method in which the 5′ end of anoligonucleotide is phosphorylated by ³²P using T4 polynucleotide kinase;

and a method (the random primed method or the like) in which substratebases labeled with an isotope such as ³²P, a fluorescent dye, or biotinare incorporated into an oligonucleotide, using a DNA polymerase such asKlenow enzyme and using random hexamer oligonucleotides and the like asa primer.

The oligonucleotides of the present invention can be produced, forexample, with a commercially available oligonucleotide synthesizer. Theprobes can also be produced as double-stranded DNA fragments obtained byrestriction enzyme treatment or the like.

Further, the present invention provides uses of DNAs suppressing Pi21gene expression and vectors comprising the DNAs. That is, the presentinvention relates to agents for increasing field resistance to blast inplants, which comprise any one of a DNA suppressing Pi21 gene expressionand a vector comprising the DNA as an active ingredient. Moreover, thepresent invention relates to uses of DNAs suppressing Pi21 geneexpression and vectors comprising the DNAs, for preparing agents forincreasing field resistance to blast in plants.

The agents for increasing field resistance to blast in plants of thepresent invention may include, for example, sterilized water,physiological saline, vegetable oil, surfactants, lipids, solubilizingagents, buffers, and preservatives, if needed, in addition to activeingredients, i.e., the oligonucleotides.

The present invention also provides molecular markers linked to thesusceptibility gene Pi21 or the resistance gene pi21.

The term “molecular marker” in the present invention means a DNA regionwhich is genetically linked to the Pi21 gene or the pi21 gene anddistinguishable from other DNA regions.

In general, when the map distance between a gene and a molecular markerexpressed with cM unit is shorter, the molecular marker is locatedcloser to the gene. Such a molecular marker is highly useful because itwill be inherited together with the gene. pi21 was shown to be locatedbetween the marker “Pa102484” and the marker “P702D3_(—)#12” (FIG. 2 c).Accordingly, in the methods of the present invention, among themolecular markers described in FIG. 2 c, the above two markers and themarkers located between the two markers (“P702D03_(—)#38”,“P702D03_(—)#79”, “P702D03_(—)#80”) are preferable. Among them,“”P702D03_(—)#79″ is an especially preferable marker and can beexemplified as the DNA of SEQ ID NO: 7 or 23 (linked to thesusceptibility gene Pi21), or SEQ ID NO: 10 (linked to the resistancegene pi21).

The molecular markers of the present invention include Sequence TaggedSite (STS) markers. The term “STS marker” refers to a DNA region whichcan be used to judge the presence or absence of polymorphism of asequence tagged site (STS) on a DNA, and the term “STS” refers to aspecific sequence site at a particular position of a DNA. Thepolymorphism of STS can be detected as the presence or absence of bandsor the difference in band position, by amplifying a DNA regioncomprising a specific sequence site with a nucleic acid amplificationmethod such as the PCR method, and then subjecting the amplificationproducts to agarose or polyacrylamide gel electrophoresis.

When a STS marker is used as a molecular marker of the presentinvention, the identification methods of the present invention can becarried out as follows, for example. First,

DNA samples are prepared by a well-known method from a test rice plantand from a rice plant having field resistance to blast. Next, a nucleicacid amplification reaction (for example, the PCR method) is carried outby using the prepared DNAs as a template and using primer DNAs. Thesizes of the amplified DNA fragments are compared between the test riceplant and a marker linked to the gene for field resistance to blast (forexample, the marker of SEQ ID NO: 10) by electrophoresis or the like,and when they show the same genotype, it is judged that the test planthas field resistance to blast.

One skilled in the art can appropriately design optimal primer DNAs usedfor the identification methods of the present invention, considering thesequence information on various molecular markers. Usually, theabove-mentioned primers are a primer set consisting of a pair of primerswhich are designed to sandwich a nucleotide sequence that specificallyexists in rice and is linked to the Pi21 gene or the pi21 gene, foramplifying the nucleotide sequence.

Specifically, primer sets for STS markers can include the following, forexample:

(a) a primer set consisting of primers (SEQ ID NO: 8)5′-AGA AGG TGG AGT ACG ACG TGA AGA-3′ and (SEQ ID NO: 9)5′-AGT TTA GTG AGC CTC TCC ACG ATT A-3′,(b) a primer set consisting of primers (SEQ ID NO: 16)5′-GTA CGA CGT GAA GAA CAA CAG G-3′ and (SEQ ID NO: 17)5′-GCT TGG GCT TGC AGT CC-3′, and (c) a primer set consisting of primers(SEQ ID NO: 26) 5′-GAT CCT CAT CGT CGA CGT CTG GC-3′ and (SEQ ID NO: 27)5′-AGG GTA CGG CAC CAG CTT G-3′.The presence of field resistance to blast in the test rice plant can bejudged by comparing the information characterized by the DNA sequencesamplified using these primer sets with the molecular markers of thepresent invention

Besides the above-mentioned primers, one skilled in the art can produceprimer sets having a similar function utilizing the nucleotide sequenceof SEQ ID NO: 1, 4, or 20. The primers of the present invention alsocomprise such primers.

One skilled in the art can produce PCR primers of the present inventionusing, for example, an automatic oligonucleotide synthesizer. Oneskilled in the art can also perform the methods of the present inventionby using a known polymorphism detection method such as thebelow-mentioned PCR-SSCP method using the above PCR primers, or thelike.

When molecular markers of the present invention are located in exons ofa genomic DNA, it is also possible to utilize RT-PCR using mRNAs as atemplate. By using the Taqman (a quantitative PCR detection) system(Roche), the presence or absence of amplification products can bedetected by fluorescence. Since this system does not needelectrophoresis, it enables one to carry out the identification methodsof the present invention in a short time.

The present invention further provides methods for judging that the testplants have field resistance to blast when the molecular weight or thenucleotide sequence is consistent with that of the pi21 gene, whichmethods comprise the following steps (a) to (c):

-   (a) preparing DNA samples from test plants;-   (b) amplifying the region of Pi21 gene or pi21 gene from the DNA    samples; and-   (c) comparing the molecular weight or the nucleotide sequence of the    amplified DNA fragments with that of the pi21 gene.

One skilled in the art can prepare (extract) the above-mentioned DNAsamples of the present invention by known methods. Preferablepreparation methods include, for example, a method for extracting DNAsusing the CTAB method.

The DNA samples used in the identification methods of the presentinvention are not particularly limited; however, genomic DNAs extractedfrom a test plant, rice, are usually used.

The source for the genomic DNA extraction is not particularly limited,and the DNAs can be extracted from any tissue of rice. They can beextracted, for example, from a panicle, leaf, root, stem, seed,endosperm portion, bran, or embryo.

In the methods of the present invention for identifying field resistanceto blast in plants, a nucleic acid amplification reaction (for example,the PCR method) is subsequently carried out by using the prepared DNAsas a template and using primer DNAs. The amplified DNA fragments arecleaved by restriction enzymes, and the sizes of the cleaved DNAfragments are compared between the test plants and plants having fieldresistance to blast, by electrophoresis or the like. When the molecularweight or the nucleotide sequence is consistent with that of thecompared plants, the test plants are judged to have field resistance toblast. “Plants having field resistance to blast” include Owarihatamochidescribed in Examples, but are not limited thereto.

In the methods of the present invention for judging that test plantshave field resistance to blast, the term “consistent with” means thatthe molecular weight or the nucleotide sequence of both genes of anallele is consistent with that of a plant having field resistance toblast, or that the deduced amino acid sequence of such genes isconsistent with that of the plant.

Accordingly, when the molecular weight, nucleotide sequence, or deducedamino acid sequence of one gene of an allele differs from that of aplant having field resistance to blast, but that of the other gene ofthe allele is the same as that of the plant, such a case is not includedin the term “consistent with”.

The above-mentioned electrophoresis analysis may be conducted accordingto a conventional method. For example, electrophoresis is carried out byapplying voltage in an agarose or polyacrylamide gel, and the separatedDNA pattern is analyzed.

The present invention also provides methods for judging that the testplants have field resistance to blast when the mobility on the gel isconsistent with that of the pi21 gene, which methods comprise thefollowing steps (a) to (d):

-   (a) preparing DNA samples from test plants;-   (b) amplifying the region of the Pi21 gene or the pi21 gene from the    DNA samples;-   (c) separating the amplified double-stranded DNAs on a    non-denaturating gel; and-   (d) comparing the mobility of the separated double-stranded DNAs on    the gel with that of the pi21 gene.

The present invention further provides methods for judging that the testplants have field resistance to blast when the mobility on the gel isconsistent with that of the pi21 gene, which methods comprise thefollowing steps (a) to (e):

-   (a) preparing DNA samples from test plants;-   (b) amplifying the region of the Pi21 gene or the pi21 gene from the    DNA samples;-   (c) dissociating the amplified DNAs into single-stranded DNAs;-   (d) separating the dissociated single-stranded DNAs on a    non-denaturating gel; and-   (e) comparing the mobility of the separated single-stranded DNAs on    the gel with that of the pi21 gene.

The above methods include the PCR-SSCP (single-strand conformationpolymorphism) method (“Cloning and polymerase chainreaction-single-strand conformation polymorphism analysis of anonymousAlu repeats on chromosome 11.” Genomics 1992, Jan. 1, 12(1): 139-146;“Detection of p53 gene mutations in human brain tumors by single-strandconformation polymorphism analysis of polymerase chain reactionproducts.” Oncogene 1991, Aug. 1; 6(8): 1313-1318; “Multiplefluorescence-based PCR-SSCP analysis with postlabeling.” PCR MethodsAppl. 1995, Apr. 1; 4(5): 275-282). This method is particularlypreferable for screening many DNA samples, since it has advantages suchas comparative simplicity of operation and a small amount of requiredtest sample. The principle of the method is as follows. Asingle-stranded DNA dissociated from a double-stranded DNA fragmentforms a unique higher conformation, depending on the respectivenucleotide sequence. After electrophoresis on a polyacrylamide gelwithout a denaturant, complementary single-stranded DNAs having the samechain length shift to different positions in accordance with thedifference of the respective higher conformations. The conformation of asingle-stranded DNA changes even by a substitution of one base, whichchange results in a different mobility on polyacrylamide gelelectrophoresis. Accordingly, the presence of a mutation in a DNAfragment due to a point mutation, deletion, insertion and such can bedetected by detecting the changes in the mobility.

More specifically, a region comprising a target site of the Pi21 gene orthe pi21 gene is first amplified by the PCR method or the like.Preferably, a region to be amplified is about 100 by to 600 bp inlength. In amplifying gene fragments by PCR, DNA fragments to besynthesized can be labeled by using primers labeled with an isotope suchas ³²P, a fluorescent dye, biotin and so on, or by adding substratenucleotides labeled with an isotope such as ³²P, a fluorescent dye,biotin and so on, to the PCR reaction solution. Alternatively, thesynthesized DNA fragments can be labeled after the PCR reaction byadding substrate nucleotides labeled with an isotope such as ³²P, afluorescent dye, biotin and so on using the Klenow enzyme and such. TheDNA fragments thus obtained are electrophoresed in the form of a doublestrand on a polyacrylamide gel without a denaturant such as urea.Alternatively, such DNA fragments may be denatured by heating and thelike, and then subjected to electrophoresis on a polyacrylamide gelwithout a denaturant such as urea. The conditions for separating DNAfragments can be ameliorated by adding appropriate amounts (about 5% to10%) of glycerol to the polyacrylamide gel. Further, although theelectrophoresis conditions varies depending on the properties ofrespective DNA fragments, it is usually carried out at room temperature(20° C. to 25° C.). When a preferable separation cannot be achieved, atemperature to achieve the optimal mobility is selected fromtemperatures between 4° C. and 30° C. After the electrophoresis, themobility of the DNA fragments is detected by autoradiography using X-rayfilms, a scanner for detecting fluorescence and the like, to analyze theresult. When bands with different mobility are detected, the presence ofa mutation can be confirmed by directly excising the bands from the gel,amplifying them again by PCR, and directly sequencing the amplifiedfragments. Even when labeled DNAs are not used, the bands can also bedetected by staining the gel after electrophoresis with ethidiumbromide, silver and such.

The present invention further provides methods for judging that the testplants have field resistance to blast when the sizes of the detected DNAfragments are consistent with that of the pi21 gene, which methodscomprise the following steps (a) to (e):

-   (a) preparing DNA samples from test plants;-   (b) amplifying the region of the Pi21 gene or the pi21 gene from the    DNA samples;-   (c) cleaving the prepared DNA samples with restriction enzymes;-   (d) separating the DNA fragments according to their sizes; and-   (e) comparing the sizes of the detected DNA fragments with that of    the pi21 gene.

The above methods include the RFLP method using Restriction FragmentLength Polymorphism (RFLP) and the PCR-RFLP method. Restriction enzymesare generally used as enzymes to cleave DNAs. Specifically, when anucleotide addition or deletion exists in the recognition site of arestriction enzyme, or when a nucleotide insertion or deletion exists ina DNA fragment generated by a restriction enzyme treatment, the sizes ofthe fragments generated after the restriction enzyme treatment differbetween plants susceptible to blast and plants having field resistanceto blast. The portion comprising such a mutation site is amplified bythe PCR method, and then treated with each restriction enzyme to detectthe polymorphic site as a difference in the mobility of bands byelectrophoresis. Alternatively, a polymorphic site can be detected bytreating chromosomal DNAs with such a restriction enzyme, subjecting thefragments to electrophoresis, and then carrying out Southern blottingwith a probe DNA. The restriction enzymes to be used can beappropriately selected in accordance with respective mutation sites. Inthis method, Southern blotting can be performed not only on genomic DNAsbut also on cDNAs which are synthesized by a reverse transcriptase fromRNAs prepared from subjects and then directly cleaved with restrictionenzymes. Alternatively, a part or whole of the Pi21 gene or the pi21gene can be amplified by PCR using such cDNAs as a template, and cleavedwith restriction enzymes, and then the difference in mobility can beexamined.

The present invention provides methods for judging that the test plantshave field resistance to blast when the mobility on the gel isconsistent with that of the pi21 gene, which methods comprise thefollowing steps (a) to (d):

-   (a) preparing DNA samples from test plants;-   (b) amplifying the region of the Pi21 gene or the pi21 gene from the    DNA samples;-   (c) separating the amplified DNAs on a gel with a gradually    increasing concentration of a DNA denaturant; and-   (d) comparing the mobility of the separated DNAs on the gel with    that of the pi21 gene.

The denaturant gradient gel electrophoresis method (DGGE method) can beexemplified as one of such methods. A region comprising a target site ofthe Pi21 gene or the pi21 gene is amplified by the PCR method and thelike using a primer of the present invention and such; the resultingproducts are electrophoresed on a polyacrylamide gel with a graduallyincreasing concentration of a denaturant such as urea; and the result iscompared with that of a healthy subject. A polymorphism can beidentified by detecting the difference in mobility of the DNA fragments,since the mobility rate of fragments with mutations decreasesdrastically as the DNA fragments become single-stranded DNAs at lowerdenaturant concentration points.

In addition to the above-mentioned methods, the Allele SpecificOligonucleotide (ASO) hybridization method can be used. Anoligonucleotide comprising a nucleotide sequence where a polymorphism ispredicted to exist, is prepared, and is subjected to hybridization witha DNA sample. When a polymorphic nucleotide different from theoligonucleotide exists in the sample DNA used for hybridization, theefficiency of hybridization is reduced. The reduction of thehybridization efficiency can be detected by the Southern blottingmethod; methods which utilize specific fluorescent reagents that have acharacteristic to quench by intercalation into a gap of a hybrid; andthe like.

Furthermore, the detection may be conducted by the ribonuclease Amismatch truncation method. Specifically, a region comprising a targetsite of the Pi21 gene or the pi21 gene is amplified by the PCR methodand the like, and the amplified products are hybridized with labeledRNAs which are prepared from healthy-type cDNAs and such incorporatedinto a plasmid vector and the like. Since the hybrid forms a singlestrand conformation in a portion comprising a nucleotide different fromthe healthy-type, a polymorphism can be detected by cleaving thisportion with ribonuclease A and then performing autoradiography and thelike.

In the present invention, the term “test plant” is not particularlylimited, but includes all plants that can be infected with the blastfungus. A preferable example is rice. Every variety of rice can be usedwithout any particular restriction, such as Indica or Japonica ricevarieties, and Indica-Japonica hybrid varieties/lines, wild rice, orcultivar-wild rice hybrid or crossbred varieties.

The present invention also provides methods for judging field resistanceto blast in rice by using as an indicator a molecular marker which islinked to the pi21 gene and comprises at least the DNA of SEQ ID NO: 7,10, or 23. Preferable molecular markers of the present invention include“P702D03_(—)#38”, “P702D03_(—)#79”, and “P702D03_(—)#80”, as mentionedabove. Among them, “P702D03_(—)#79” is an especially preferable marker,and it may be the DNA of SEQ ID NO: 7 or 23 (linked to thesusceptibility gene Pi21) or SEQ ID NO: 10 (linked to the resistancegene pi21), for example. The identification methods of the presentinvention use as an indicator at least “P702D03_(—)#79” among thesemolecular markers. Therefore, in the identification methods of thepresent invention, “P702D03_(—#79)” may be used alone or in combinationwith other markers. The combinations of “P702D03_(—)#79” with othermarkers include the combination with “P702D03_(—)#38”, combination with“P702D03_(—)#80”, and combinations with any other markers.

In the identification methods of the present invention, field resistanceto blast in test rice plants can be judged specifically and efficientlyby examining whether they comprise a molecular marker linked to the pi21gene. In the judging methods of the present invention, when a desiredrice plant to be judged for having field resistance to blast or notcomprises the nucleotide sequence of SEQ ID NO: 10, the test rice plantis judged to have field resistance to blast. When the test rice plantdoes not comprise the nucleotide sequence of SEQ ID NO: 10 (when itcomprises the nucleotide sequence of SEQ ID NO: 7 or 23), it is judgedto be susceptible to blast.

Molecular markers in test rice plants can be compared with those of thepresent invention not only for the DNA sequences of molecular markers,but also for the information characterized by the DNA sequences. Theinformation characterized by the DNA sequences of molecular markersincludes information about the molecular weight of the molecular markersand about the presence or absence of a mutation site and polymorphicsite comprised in the molecular markers. One skilled in the art canidentify polymorphic sites (deletion sites and single base-substitutionsites) by comparing the nucleotide sequence of SEQ ID NO: 10 with thatof SEQ ID NO: 7 or 23 using known methods. The judging methods of thepresent invention can also be performed by detecting such information onthe presence or absence of a mutation site or polymorphic site comprisedin molecular markers.

The above information on the presence or absence of a mutation site orpolymorphic site can be detected by using primers which can amplify aregion comprising a mutation site or polymorphic site, or by using aprobe (for example, the DNA comprising the whole or a part of thenucleotide sequence of SEQ ID NO: 18, 19, or 25) which can hybridize toa mutation site or polymorphic site, as well as by directly determiningthe sequences.

By using the judging methods of the present invention, it becomespossible to select at an early stage plants (for example, rice) to beidentified as having field resistance to blast. Specifically, thepresent invention provides methods for selecting plants having fieldresistance to blast, which comprise the following steps (a) and (b):

-   (a) producing varieties in which plants (for example, rice) having    field resistance to blast have been crossed with plants (for    example, rice) having arbitrary functions;-   (b) judging whether the plants obtained in step (a) have field    resistance to blast by the methods herein described for judging    whether test plants have field resistance to blast.

The term “plant” is not particularly limited in the present invention,but preferably refers to rice. Specific examples of rice are asmentioned above.

By using the selection methods of the present invention, it becomespossible to select at an early stage plants (for example, rice) to beidentified as having field resistance to blast. The present inventionalso provides such methods for selecting at an early stage plants to beidentified as having field resistance to blast. As used herein, the term“early stage” refers to, for example, the state before heading of rice,and preferably the state immediately after germination. By using theselection methods of the present invention, it becomes possible to breedvarieties having field resistance to blast in a shorter period thanbefore.

The present invention relates to methods of screening for agents toprevent or ameliorate blast in plants. The first embodiment of thescreening methods of the present invention includes methods of screeningfor agents to prevent or ameliorate blast in plants, which comprise thefollowing steps (a) to (c):

-   (a) contacting test compounds with a Pi21 gene transcription    product;-   (b) detecting the binding of the test compounds to the Pi21 gene    transcription product; and-   (c) selecting test compounds that bind to the Pi21 gene    transcription product.

In the first embodiment, test compounds are first contacted with thePi21 gene transcription product. “Pi21 gene transcription product” inthe screening methods of the present invention includes not only thePi21 gene transcription product, but also the translation producttranslated from the transcription product.

The “test compounds” in the methods of the present invention are notparticularly limited, and include, for example, single compounds such asnatural compounds, organic compounds, inorganic compounds, proteins, andpeptides; as well as compound libraries, expression products of genelibraries, cell extracts, cell culture supernatants, products offermentation microorganisms, marine organism extracts, plant extracts,prokaryotic cell extracts, unicellular eukaryote extracts, and animalcell extracts. If needed, the above test compounds can be appropriatelylabeled before use. Labels include, for example, radiolabels andfluorescent labels.

In the present invention, “contacting” is carried out as follows. Forexample, if the Pi21 gene transcription product is in a purified state,the contact can be carried out by adding test compounds to the purifiedpreparation. If the transcription product is in the state expressed incells, or in the state expressed in cell extracts, the contact can becarried out by adding test compounds to the cell cultures or to the cellextracts, respectively. The cells in the present invention are notparticularly limited, but cells derived from plants including rice arepreferable. When the test compounds are proteins, the contact can alsobe carried out, for example, by introducing vectors comprising the DNAsencoding the proteins into cells expressing the Pi21 gene, or by addingthe vectors to cell extracts in which the Pi21 gene is expressed.Further, for example, two hybrid methods using yeast or animal cells canbe utilized.

In the first embodiment, the binding between the above-mentioned Pi21gene transcription product and test compounds is subsequently detected.Detection or measurement of the binding between proteins can be carriedout by using, for example, labels attached to the proteins. The types oflabels include, fluorescent labels and radiolabels, for example. Thebinding can also be measured by known methods such as the yeast twohybrid method and the method using BIACORE. In the present methods, thetest compounds bound to the above-mentioned biosynthesis enzyme are thenselected. Among the selected test compounds, agents for preventing orameliorating blast in plants are included. The selected test compoundsmay be used as test compounds in the following screenings.

In addition, the second embodiment of the screening methods of thepresent invention provides methods of screening for agents to prevent orameliorate blast in plants, which comprise the following steps (a) to(c):

-   (a) contacting test compounds with cells collected from plants;-   (b) measuring the expression level of thePi21 gene transcription    product; and-   (c) selecting the test compounds that decrease the expression level    of the transcription product as compared to when the test compounds    are not contacted with the cells.

In the second embodiment, test compounds are first contacted with cellscollected from plants. As used herein, the term “cells collected from aplant” may be an arbitrary plant clearly having a blast susceptibilitygene. The terms “test compound” and “contacting” refer to the same asmentioned above.

In the second embodiment, the expression level of the “Pi21 protein” issubsequently measured. The expression level of the Pi21 protein can bemeasured by methods known to one skilled in the art. For example, mRNAencoding the Pi21 protein is extracted according to a conventionalmethod, and the transcription level of the Pi21 gene can be measured byperforming the Northern hybridization method or the RT-PCR method usingthis mRNA as a template. Further, the expression level of the Pi21protein can be measured using DNA array techniques.

The translation level of the gene can also be measured by collectingfractions comprising the Pi21 protein in accordance with a usual method,and detecting the expression of the Pi21 protein by electrophoresis suchas SDS-PAGE. The translation level of the gene can also be measured byperforming the Western blotting method using an antibody against thePi21 protein to detect the expression of the Pi21 protein.

The antibodies used for detection of the Pi21 protein are notparticularly limited, as long as they can detect the Pi21 protein. Bothmonoclonal antibodies and polyclonal antibodies can be used, forexample. The antibodies can be prepared as mentioned above, by methodsknown to one skilled in the art.

In the second embodiment, next, when the expression level of the Pi21protein decreases compared to when the test compounds are not contacted,the test compounds are selected as agents to prevent or ameliorate blastin plants.

The third embodiment of the screening methods of the present inventionprovides methods of screening for agents to prevent or ameliorate blastin plants, which comprise the following steps (a) to (d):

-   (a) providing cells or cell extracts comprising DNAs in which a    reporter gene is operably linked downstream of the promoter region    of the Pi21 gene;-   (b) contacting test compounds with cells or the cell extracts;-   (c) measuring the expression level of the reporter gene in the cells    or the cell extracts; and-   (d) selecting test compounds that decrease the expression level of    the reporter gene as compared to when the test compounds are not    contacted.

In the third embodiment, cells or cell extracts comprising DNAs in whicha reporter gene is operably linked downstream of the promoter region ofthe Pi21 gene, are first provided.

In the third embodiment, the term “operably linked” means that thepromoter region of the Pi21 gene and a reporter gene are connected toeach other so that the reporter gene expression can be induced bybinding of a transcription factor to the promoter region of the Pi21gene. Therefore, the term “operably linked” also inlcudes such caseswhere a reporter gene is connected to another gene and produces a fusedprotein with another gene product, as long as expression of the fusedprotein is induced by binding of a transcription factor to the promoterregion of the Pi21 gene.

The reporter gene is not particularly limited, so long as its expressioncan be detected. For example, reporter genes generally used by thoseskilled in the art include the CAT gene, lacZ gene, luciferase gene,β-glucuronidase gene (GUS), and GFP gene.

In the third embodiment, the above-mentioned cells or cell extracts aresubsequently contacted with the test compounds. Then, the expressionlevel of the reporter gene in the cells or the cell extracts ismeasured. The terms “test compound” and “contacting” refer to the sameas mentioned above.

The expression level of the reporter gene can be determined usingmethods known to those skilled in the art, according to the type ofreporter gene. For example, when using the CAT gene as the reportergene, the CAT gene expression level can be determined by measuring theacetylation of chloramphenicol, caused by the CAT gene product. When thelacZ gene is used as the reporter gene, its expression level can bedetermined by analyzing the colouring of a dye compound due to thecatalytic action of the gene expression product. The expression level ofthe luciferase gene as a reporter can be determined by measuring thefluorescence of a fluorescent compound, caused by the catalytic actionof the luciferase gene expression product. The expression level of theβ-glucuronidase (GUS) gene can be determined by analyzing the coloringof 5-bromo-4-chloro-3-indolyl-β-glucuronide (X-Gluc) or the luminescenceof Glucuron (ICN), caused by the catalytic action of the GUS geneexpression product. The expression level of the GFP gene can bedetermined by measuring fluorescence due to the GFP protein.

Next in the third embodiment, if the expression level of theabove-mentioned genes decrease compared to when the test compounds arenot contacted, the test compounds are selected as agents to prevent orameliorate blast in plants.

The fourth embodiment of the screening methods of the present inventionprovides methods of screening for agents to prevent or ameliorate blastin plants, which comprise the following steps (a) to (c):

-   (a) regenerating transformed plants from transformed plant cells    into which the Pi21 gene has been introduced;-   (b) contacting the blast fungus and test compounds with the    transformed plants; and-   (c) selecting test compounds that suppress blast in the transformed    plants as compared to when the test compounds are not contacted.

In the fourth embodiment, transformed plants are first regenerated fromtransformed plant cells comprising the Pi21 gene. The transformed plantscan be regenerated as mentioned above, by a method known to one skilledin the art.

In the fourth embodiment, next, the blast fungus and test compounds arecontacted with the transformed plants regenerated in step (a). The terms“blast fungus” and “test compound” are the same as mentioned above. Anexample of “contacting” is a method for directly spraying a testcompound on a plant using a sprayer. However, “contacting” in the fourthembodiment is not limited thereto, but includes any method, as long asplants and test compounds can physically contact. The contact of thepresent invention may be performed by contacting test compounds withtransformed plants infected with the blast fungus, or by infecting withthe blast fungus transformed plants which have contacted with testcompounds.

In the fourth embodiment, next, test compounds that suppress blast intransformed plants as compared to when test compounds are not contacted,are selected. Whether blast is suppressed or not can be determined byusing as an indicator a phenotype of the transformed plants. Thephenotypes of the transformed plants are not particularly limited, butinclude discoloring and necrotizing of an entire part of a plant, or aportion of it. Moreover, suppression of blast in the transformed plantsincludes not only complete suppression but also partial suppression.

The present invention also relates to kits for use in theabove-described screening methods. Such kits can comprise materials usedat the detection step and/or measurement step in the above-describedscreening methods. For example, such materials can include probes,primers, antibodies, and stain solutions, which are necessary formeasuring Pi21 gene expression level. In addition, the kits may comprisedistilled water, salts, buffer solutions, protein stabilizers,preservatives and the like.

All prior art references cited in the present specification areincorporated herein by reference.

EXAMPLES

Hereinafter, the present invention will be specifically described withreference to Examples, but it is not construed as being limited thereto.

Example 1 Genetic Mapping

A detailed linkage analysis of the pi21 region was conducted using alarge-scale segregating population indispensable for map-based cloning.As the population for linkage analyses, 72 samples of the BC1F2population were used. This BC1F2 population was obtained by continuouslybackcrossing the paddy rice variety Nipponbare or Aichi Asahi (FIG. 1)comprising the susceptibility allele Pi21 that does not suppress blotchprogression with the Japanese upland rice variety Owarihatamochicomprising the resistance allele pi21 that suppresses blotchprogression. As a result of linkage analysis with RFLP markers, it wasfound that the pi21 gene locus is located between the RFLP markers G271and G317 (FIG. 2A).

In order to create a more accurate genetic map of the pi21 region, atotal of 1014 samples including the above-mentioned crossbred 229samples and 643 samples of the progeny BC1F4 population were used toselect 27 samples with chromosomal recombination near the pi21 locus, byusing the RFLP markers RA3591 and 13S1 located on both sides of the pi21locus (FIG. 2B). Furthermore, a search was carried out using 2703samples of F2 population, which was obtained by crossing a line havingthe genetic background of Japanese paddy rice variety and thesusceptibility allele from the Indian paddy rice variety Kasalath with aline having the resistance allele from Owarihatamochi. 24 samples withchromosome recombination near the pi21 locus were selected using the PCRmarkers 14T1 and 4S1 located on both sides of the pi21 locus.Furthermore, using those samples, a detailed linkage map was createdutilizing the DNA markers produced in the following procedures.

Example 2 Alignment of P1-Derived Artificial Chromosome (PAC) Clones inthe pi21 Gene Region

Using the alignment map of Nipponbare PAC clones produced in the ricegenome research program, PAC clones comprising the DNA markers RA3591and C975 nucleotide sequences positioned near the pi21 gene locus wereidentified (FIG. 2C). Furthermore, terminal fragments of the identifiedPAC clones P479G02, P415D09, P473G08, P703E11, P434F09, P702D03,P419B08, P472G09, and P502G01 were isolated by the cassette method, andthe identified PAC clones were aligned. As a result, it was found thatthe PAC clones P032D02, P678A02, P405D12, P689F04, P479G02, P415D09,P473G08, P703E11, P434F09, and P702D03 comprise the pi21 gene region(FIG. 2C).

Example 3 Narrowing the Candidate Region of the pi21 Gene

Terminal fragments of the PAC clones aligned in the pi21 region werecloned, and the obtained clones are used as new RFLP markers or CAPSmarkers to create a detailed genetic map. As a result, it was found thatthe pi21 gene locus exists in the genomic region sandwiched between theSSCP marker Pa102484 and the SNP marker P702D3_(—)#12. Accordingly, itwas revealed that the pi21 gene locus is located in the genomic regionof about 25 kb sandwiched by the two markers (FIG. 2D).

[Example 4 ] Identification of the Candidate Gene Region by NucleotideSequence Analysis

The nucleotide sequence of the PAC clone P702D03 considered to comprisethe pi21 gene was determined, and the nucleotide sequence of 25 kbcandidate genomic region in the resistant variety Owarihatamochi and thesusceptible varieties Aichi Asahi and Kasalath were analyzed. Thenucleotide sequence was analyzed by using the DNA fragments which wereamplified from the above-mentioned three varieties with primers designedutilizing the sequence of the candidate region in Nipponbare and byusing the dye-terminator method. The candidate region was furthernarrowed using the nucleotide polymorphism information in the candidategene region identified by linkage analysis. As a result, the pi21 genewas shown to co-segregate with the STS marker P702D03_(—)#79 (primers:5′-AGA AGG TGG AGT ACG ACG TGA AGA-3′ (SEQ ID NO: 8) and AGT TTA GTG AGCCTC TCC ACG ATT A-3′ (SEQ ID NO: 9)), and one recombinant was detectedbetween the SNP markers P702D03_(—)#38 (primers: TTT TCC TGA GAA ATT TGTAAA GA-3′ (SEQ ID NO: 12) and CGT CGA CGA TGA GGA TCT-3′ (SEQ ID NO:13)) and P702D03_(—)#80 (primers: 5′-CTC CCA ATG TGT TTA GCA TC-3′(SEQID NO: 14) and 5′-CAA CCA TAT GTC CCT AAG GAT-3′ (SEQ ID NO: 15)),respectively. These results showed that the pi21 gene is located in thegenomic region of about 1.8 kb sandwiched between the SNP markersP702D03_(—)#38 and P702D03_(—)#80 (FIG. 2D).

The nucleotide sequence of the isolated Pi21 gene derived from rice(Oryza sativa L, varieties Aichi Asahi and Nipponbare) is shown in SEQID NO: 1, the nucleotide sequence of its cDNA is shown in SEQ ID NO: 2,and the amino acid sequence of the protein (“the Pi21 protein”) encodedby the cDNA is shown in SEQ ID NO: 3. In addition, the nucleotidesequence of the Pi21 gene derived from Kasalath, corresponding to thePi21 gene of Aichi Asahi and Nipponbare, is shown in SEQ ID NO: 20, thenucleotide sequence of its cDNA is shown in SEQ ID NO: 21, and the aminoacid sequence of the protein (“″the Pi21 protein”) encoded by the cDNAis shown in SEQ ID NO: 22.

Example 5 Nucleotide Sequence Analysis of the pi21 Candidate Gene

When a gene prediction and similarity search were carried out for thesequence of the 1.8 kb candidate genomic region of the varietyNipponbare, full-length cDNA clones of Nipponbare (AK106153, AK070581,and AK072320) were discovered. However, no similar genes were present inArabidopsis or the like, and thus the function of the gene could not bepredicted from homology. Nevertheless, a metal-binding site at aposition about 10 amino acids away from the predicted translationinitiation site in this gene brings to mind a gene reported inArabidopsis (Hirayama et al., 1999 Cell) with the function of achaperone which carries a metal in the ethylene signaling system. Sincesensibility to ethylene in the near-isogenic line AA-pi21 is actuallychanged compared to Aichi Asahi, the site may have a similar function.Primers which can amplify the corresponding part were designed using thealready obtained nucleotide sequence information of Nipponbare, and thenucleotide sequences of the genomic PCR and RT-PCR products of thesusceptible varieties Nipponbare and Aichi Asahi were compared withthose of the resistant variety Owarihatamochi. As a result, DNAmutations were found at two sites in the exon region of the gene in theresistant variety compared to the susceptible varieties. In theresistant variety, deletions of 7 amino acids and 16 amino acids werefound relative to the susceptible varieties, and these mutations werethought to be associated with blotch progression in blast that hadinfected (FIG. 3).

The nucleotide sequence of the isolated pi21 gene is shown in SEQ ID NO:4, the nucleotide sequence of its cDNA is shown in SEQ ID NO: 5, and theamino acid sequence of the protein encoded by the cDNA (“the pi21protein”) is shown in SEQ ID NO: 6.

Example 6 Identification of the Function of the Candidate Gene byTransformation

(1) Introduction of the Susceptibility Gene into AA-pi21

An XbaI 4.7 kb fragment of the genomic region including 5′ upstreampredicted promoter region of the susceptible variety Nipponbare,identified as a candidate of the pi21 gene, was incorporated into thevector pPZP2H-lac that can be transformed through Agrobacterium.Transformation was carried out by the method of Toki (Plant Mol. Biol.Rep. 15: 16-21, 1997) using a vector into which this fragment had beenintroduced and a vector alone. As the line to be transformed, the pi21near-isogenic line AA-pi21 was used. 36 hygromycin-resistant organismswere obtained from the vector into which the XbaI 4.7 kb fragment hadbeen introduced, and 12 hygromycin-resistant organisms were obtainedfrom the vector alone. Whether the introduced region was incorporated ornot, was investigated by the PCR method using primers (sense strand:5′-GTA CGA CGT GAA GAA CAA CAG G-3′ (SEQ ID NO: 16)) and (antisensestrand: 5′-GCT TGG GCT TGC AGT CC 3′ (SEQ ID NO: 17)) that were specificto the candidate gene. As a result, it was found that the candidate genewas incorporated into all the transformants. These organisms were grownin an isolated greenhouse, and the blast fungus (race 007) wasinoculated into the inbred line progenies. As a result, in the organismsinto which the vector alone was introduced and the T1 organisms intowhich the introduced gene was not delivered due to segregation, blotchprogression caused by blast was suppressed as shown in the near-isogenicline AA-pi21, compared to the susceptible variety Aichi Asahi. Incontrast, in the T1 organisms into which the candidate gene had beenintroduced, blotches progressed more extensively (FIG. 4). Especially,the degree of sensibility was increased in the lines having a high copynumber of the introduced gene.

(2) Introduction of the Resistance Gene to a Susceptible Variety

On the other hand, the XbaI 4.7 kb fragment of the resistant varietyOwarihatamochi was incorporated into the vector in the same way, and thesusceptible variety Aichi Asahi was transformed. 56 Hygromycin-resistantorganisms were obtained from the vector into which the XbaI 4.7 kbfragment had been introduced, and 24 hygromycin-resistant organisms wereobtained from the vector alone. Similarly to (1), whether the introducedregion was incorporated or not was investigated by the PCR method, andit was found that the candidate gene was incorporated into all thetransformants. These organisms were grown in an isolated greenhouse inthe same way as (1), and the blast fungus (race 007) was inoculated intothe inbred line progenies. As a result, in all of the organisms intowhich the vector alone had been introduced, T1 organisms to which theintroduced gene was not delivered, and T1 organisms into which thecandidate gene had been introduced, blast progression was observed tothe same degree as that in the susceptible variety Aichi Asahi.

(3) Identification of the Function of the Candidate Gene

From the above results, it was found that the candidate gene region fromNipponbare (XbaI 4.7 kb) has the function of promoting blotch formationin the near-isogenic line AA-pi21, and thus the candidate gene wasjudged to be the Pi21 gene.

Example 7 Mutations of the Candidate Gene in Rice

Mutations of the candidate gene were searched using 79 rice varieties inthe world. As a result, in addition to the mutation types found inNipponbare, Aichi Asahi, and Owarihatamochi, ten types of mutationshaving insertions and/or deletions in the exon region were found. Thesemutations are mainly defined by the presence or absence and the size ofan insertion/deletion at the two deletion sites found in Owarihatamochicompared to Nipponbare and Aichi Asahi. Because of the similarity to themetal molecule chaperone proposed in the ethylene signaling system ofArabidopsis thaliana, this region having no homology with known genes isexpected to bind to another molecule. Thus, the mutations in this regionmay delicately control the signaling efficiency and bring aboutfunctional alterations.

From the above results, the candidate gene narrowed down by themap-based cloning method was found to be the pi21 gene which suppressesblotch progression in rice blast disease. This achievement is the firstcase to prove the biological function of a quantitative resistance in aplant. The expression of the pi21 or the Pi21 gene was investigated byRT-PCR analysis, and each gene was found to be constitutively expressedin all the tissues of the aerial part. Therefore, it is expected thatthese genes play a fundamental role in the growth of plants. Sincechange of the copy number leads to phenotype changes, alteration of theexpression level and the tissues where the genes are expressed bypromoters can be an important factor for functional modification. Thatis, it may be possible to efficiently ameliorate disease resistance thatplants originally have, by utilizing the isolated pi21 gene or otheralleles found in the species.

INDUSTRIAL APPLICABILITY

The characteristics of the Pi21 gene are especially suitable forproducing varieties having field resistance to blast in plants. Untilnow, in order to confer plants with field resistance to blast, it wasnecessary to cross a variety that originally has field resistance andinferior characteristics with a variety that does not have fieldresistance but has many superior characteristics, and to select fromamong their progenies, plants having excellent field resistance as wellas other excellent characteristics. However, the precise evaluation offield resistance needs a lot of effort. Moreover, when the exactposition on the chromosome of the gene that confers this resistance isunclear, it is difficult to select this gene efficiently and accuratelyand to introduce it into a variety with a high practical use. In fact,this had not succeeded until now.

The present invention provides the chromosomal position and thestructure of the gene involved in field resistance. Thus it becamepossible to efficiently confer plants with field resistance. It alsobecame possible to breed varieties having resistance and highlypractical characteristics by changing the tissue specificity ofexpression and the expression level of the gene participating in fieldresistance. Accordingly, the genes of the present invention are usefulfor realizing very practical and highly safe agriculture. Moreover,plants produced by the methods of the present invention are expected,for example, to stably give a high yield when it comes to usefulagricultural plants, and also gain a new aesthetic value when it comesto ornamental plants.

1. A set of nucleic acids capable of amplifying all or a part of thenucleotide sequence of SEQ ID NO: 1, 4, or
 20. 2. The set of claim 1,comprising at least one of the following sets of nucleic acids selectedfrom the group consisting of: (a) a nucleic acid comprising thenucleotide sequence of SEQ ID NO: 8, and a nucleic acid comprising thenucleotide sequence of SEQ ID NO: 9; (b) a nucleic acid comprising thenucleotide sequence of SEQ ID NO: 16, and a nucleic acid comprising thenucleotide sequence of SEQ ID NO: 17; and (c) a nucleic acid comprisingthe nucleotide sequence of SEQ ID NO: 26, and a nucleic acid comprisingthe nucleotide sequence of SEQ ID NO:
 27. 3. An isolated nucleic acidcomprising the nucleotide sequence of SEQ ID NO: 7, 10, 18, 19, 23, or25.